PROJECT SUMMARY This proposal seeks to develop a resource for the preservation of the fruit fly, Drosophila melanogaster. This insect is a foundational model organism for biological research. Over a century of work, an enormous number of fly strains harboring different mutant alleles or transgenic constructs have been generated. However, one limitation of working with flies is that there is as yet no practical method for cryopreservation of Drosophila strains. Conventional methods of vitrifying Drosophila were developed in the early 1990s and were never widely adopted due to the difficulty in performing the protocols. This is a problem from a practical perspective since all these strains need to be individually maintained in continuous culture at substantial cost and labor, and also from a scientific perspective, since in the process of continuous culture mutations can accumulate and contamination can occur, degrading the value of these resources for future experiments. A novel approach for cryopreservation of Drosophila is proposed for this R24 resource center. Isolated embryonic nuclei, rather than intact embryos, will be cryopreserved and then nuclear transplantation via microinjection will be used to create clones derived from the cryopreserved nuclei. This approach avoids the issues associated with the impermeability of embryonic membranes that have prevented the use of conventional cryopreservation approaches that have been used with other organisms. Embryonic nuclei will be cryopreserved using a naturally inspired approach. Diverse biological systems (plants, insects, etc.) survive dehydration, drought, freezing temperatures and other stresses through the use of osmolytes. On an applied level, the proposed investigation has the potential to transform preservation of Drosophila lines by 1) preserving subcellular components (specifically nuclei) as opposed to embryos; and 2) automating much of the workflow. In the long- term, the goal of this resource center is to develop a robust and scalable protocol for cryopreservation of Drosophila, thus reducing the cost and improving the quality of long-term strain maintenance.